GSM6619765: Day 6, non-elongated; Mus musculus; RNA-Seq
The sample preparation was performed according to the instructions from 10X Genomics Single Cell Protocols (10X Genomics), with some modification. The collected organoids (100 organoids per condition) were washed twice with PBS and trypsinized with TrypLE Express (Gibco, 12605-028) for 7 min in 37°C. The organoids were dissociated into single cells by pipetting up and down with a 1000 μl pipette tip, strained using 40 μm cell strainer and collected in 1.5 μl microcentrifuge tube. Those single cells were centrifuged at 300 g for 5 min and resuspended in 1 ml of PBS with 0.04% BSA after removing the supernatant. The cell concentration was then determined. After the same centrifugation step was repeated, the cells were resuspended in PBS with 0.04% BSA to achieve a final cell concentration of 1 million cells/ml. After the trypsinization, the cells were always placed on ice. Reverse transcription and library preparation were performed using the Single Cell 3′ Reagent Kit v3 (10X Genomics), following the 10X Genomics protocol.